Method for improving the cell protection

ABSTRACT

The invention relates to a method for improving the cell protection which comprises the administration of a combination of the extracts of the plants  Vitis vinifera  and  Lycopersicon esculentum  with the vitamins C, E, beta-carotene and optionally selenium. Said combination can be used in the prevention of phatological conditions related in part to an overproduction of free radicals like aging, arteriosclerosis and cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a divisional of U.S. patentapplication Ser. No. 09/863,768, Filed on May 24, 2001, which claimsbenefit of the filing date of U.S. appl. Ser. No. 60/206,945, filed May25, 2000, the disclosures of which are incorporated herein in entiretyby reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention is related to a novel method of improvingthe cell protection, which utilises the administration of thecombination of the extracts of the plants Vitis vinifera andLycopersicon esculentum with the vitamins C, E, beta-carotene andselenium, as well as the use of such combination for treating orpreventing pathologies related to an overproduction of free radicalslike aging, arteriosclerosis and cancer.

[0004] 2. Background Art

[0005] Antioxidants act to protect components of the body against freeradical damage (Harman D.; Holliday R. and M. Meydany—Editor, “TowardsProlongation of healthy life span”, Annals of the New York Academy ofSciences, Vol. 854, 1998, New York; Stählin H. B., “The Impact ofAntioxidants on Chronic Disease in Aging and Old Age”, Int. J. Vitamin.Nutr. Res. 69, 146-149, 1999).

[0006] Several epidemiological studies support the observation that anincrease in antioxidant intake limits the clinical expression ofcoronary artery diseases and some tumours (Comstock G W; Alberg A J;Huang H Y, Wu K.; Burke A E; Hoffinan S C; Norkus E P; Gross M.; CutlerR G; Morris J S; Spate V L; Helzlsouer K J, “The risk of developing lungcancer associated with antioxidants in the blood: ascorbic acid,carotenoids, alpha-tocopherol, selenium, and total peroxyl radicalabsorbing capacity”, Cancer Epidemiol Biomarkers Prev 6, 907-916, 1997).Individuals with high dietary intakes of antioxidants may have a greaterlife expectancy.

[0007] Individual conditions, environmental stress and oxidative stressinduced by environmental and nutritional factors exert its influence ona wide variety of individuals with varying defense capacities.

[0008] In general terms, individuals with very well balanced defensemechanisms will remain free of chronic disease, but will eventuallysuccumb to the overall aging processes. Much more likely, however, isthe case where critical systems are insufficiently protected and becomeinjured. The sequence of this process will be clinically diagnosed aschronic disease.

[0009] Antioxidants as important elements in the body's defense againstoxidative stress thus have a general anti-aging property as well as aspecific disease-protective function. Since the consequences of chronicdisease are among the leading causes of disability in old age, primaryprevention by adequate antioxidative defense has not only an“anti-aging” function, but more importantly, a specificchronic-disease-preventing function. However, it becomes clear that thisis a life-long important protective mechanism: primary prevention ofage-related chronic diseases has to start early in life and needs to becontinued throughout the whole life span.

[0010] The relationship between antioxidants and chronic disease isprobably best investigated in cardiovascular disease. A strong inverserelationship between plasma antioxidant concentration, morbidity, andmortality from coronary heart disease was demonstrated. The analyses ofthe data allowed to quantify the contribution of the differentantioxidative vitamins. According to these results a sufficient intakeof vitamin E is most important, followed by vitamin C and caroteneintake.

[0011] In the analysis of a population with an overall very good vitaminE status, a strong effect of vitamin C and beta-carotene was observed.It is quite well known that other antioxidants without vitamin functionsoriginating from food, such as flavonoids, polyphenols, or lycopene havevery important antioxidative functions (Fuhrman B.; Ben-Yaish L.; AttiasJ.; Hayek T.; Avaram M., “Tomato lycopene and beta-carotene inhibitlow-density lipoprotein oxidation and this effect depends on thelipoprotein vitamin E content”, Nutr. Metab. Cardiovasc. Dis. 7,433-443, 1997; Pastori M.; Pfander H.; Boscoboinik D.; Azzi A.,“Lycopene in association with alpha-tocopherol inhibits at physiologicalconcentrations proliferation of prostate carcinoma cells”, Biochem.Biophys. Res. Commun. 250 (3), 582-585, 1998; Boehm F.; Edge R.;McGarvey D J; Truscott T G, “Beta-carotene with vitamins E and C offerssynergistic cell protection against Nox”, FEBS Lett. 436, 387-389, 1998;Watkins T. R.—Editor, “Wine, Nutritional and Therapeutic Benefits”,American Chemical Society Symposium Series 661, 1997, Washington D.C.).

[0012] In late adulthood and early old age, cancer is the leading causeof premature death. There is no question that a large majority ofcancers are the result of environmental factors challenging the specificgenetic endowment of tissue at high risk for proliferative disorders.Epidemiological evidence clearly demonstrates that a low intake of fruitand vegetables and low plasma concentrations of antioxidative vitaminscorrelated with a significantly elevated cancer morbidity and mortality.This holds true for gastrointestinal cancer and lung cancer, but alsofor prostate cancer and for breast cancer.

[0013] The neoplastic process is a highly complex sequence of events.Many potentially dangerous mutations occur continuously. Once thesegenetic changes have escaped the initial defense and repair mechanisms,these altered cells have to be eliminated by other defense mechanisms.One important mechanism is the apoptotic process. It may be thatcommitted cells in the proliferative stage are protected by antioxidantsagainst the body's own immune and apoptotic defense mechanisms. Thismight explain why antioxidants occur in higher concentration in breastcancer tissue. There is no doubt that a life-long high intake of a dietrich in fruit and vegetables and high in a variety of antioxidantssubstantially and significantly lowers the risk of malignant disordersin later life.

[0014] Primary degenerative brain disease and diseases related tocerebral vascular disturbances are the leading cause of disability inold age. Loss of autonomy, dependence and high social costs forindividuals and society are the consequences of brain dysfunction. Theimpressive gain in individual life expectancy is overshadowed by theloss of mental function in old age. Hence, prevention and treatment ofdisorders leading to mental impairment have emerged as major challengeof the modern health care system. As in the previous examples, acombination of age-related functional changes superimposed by disordersof circulation or of certain neuronal systems leading to distinctchronic diseases are being observed.

[0015] Given the importance of antioxidants in the maintenance of cellintegrity and cell function, antioxidants (vitamin E, carotenes, vitaminC) and other micronutrients from the food, e.g. polyphenols, flavonoids,lycopene, may decrease the risk of vascular disease, protect the neuronsagainst oxidative stress and thus maintain neuronal function. Severalepidemiological studies in elderly people have revealed a correlationbetween antioxidants and cognitive performance.

[0016] Other epidemiological data have shown that red wine may reducethe mortality rate from heart disease, the so called “French paradox”.An analysis conducted on 34,014 men between 1978 and 1983 in EasternFrance has shown that a moderate intake of wine (2-5 glasses of wine)was associated with a 24-31% reduction in all-cause mortality (Renaud S.C. et al., “Alcohol and Mortality in Middle-Aged Men from EasternFrance”, Epidemiology 9, 184-188, 1998).

[0017] Although many studies have been conducted with single antioxidantcompounds, the pharmacological effects of the administration of thecombination of antioxidant substances of natural origin as plantextracts of Vitis vinifera (Grape), of Lycopersicon esculentum (Tomato)with the vitamins C, E, beta-carotene and selenium have never beenstudied.

[0018] The Israeli Patent Application IL 121112 (Jun. 19, 1997)describes a synergistic mixture containing lycopene and vitamin E andits use in the prevention of LDL oxidation. The applicant also publishedthe results in a scientific paper describing that the LDL protection bylycopene exceeded the protection by β-carotene. However, this effect wasselective only to LDL's with high vitamin E content and was potentiatedwhen the carotenoids were present in combination with vitamin E (FuhrmanB.; Ben-Yaish L.; Attias J.; Hayek T.; Avaram M., loc. cit.).

[0019] The U.S. Pat. No. 5,648,377 (Jul. 15, 1997) relates to thecombination of lycopene with an extract of Vitis vinifera showing thatthe combination exerts a synergistic antioxidant action.

BRIEF SUMMARY OF THE INVENTION

[0020] The object of this invention is the surprising discovery that theadministration of the combination of the extracts of the plants Vitisvinifera and Lycopersicon esculentum combined with vitamin C, vitamin E,β-carotene and optionally selenium significantly increases, in asynergistic manner, the cell protection. The results obtained from thedescribed experimental tests show that the improvement of the cellprotection with the combination of the mentioned substances arestatistically greater than the combination of an extract of Vitisvinifera with lycopene (U.S. Pat. No. 5,648,377) or the combination ofvitamin E with lycopene (IL Patent application 121112).

[0021] The invention therefore relates to a improved method ofprotecting the cell from a disease caused by an overproduction of freeradicals, said method comprises administering a protective ortherapeutically effective amount of a composition comprising synergisticamounts of a lipophilic antioxidant and a hydrophilic antioxidant andoptionally an acceptable carrier, the improvement wherein is that saidlipophilic antioxidant is a mixture consisting essentially of vitamin E,beta carotene and an extract of Lycopersicum esculentum, and saidhydrophilic antioxidant is a mixture consisting essentially of vitamin Cand an extract of Vitis vinifera.

[0022] In another aspect, the present invention provides a method oftreating a subject suffering from a disease, which is caused by anoverproduction of free radicals, comprising the administration of atherapeutically effective amount of a composition according to thepresent invention.

[0023] In a further aspect, the present invention provides a method oftreating a subject suffering from an inflammation.

[0024] In another aspect, the method according to the present inventionis applicable to the treatment of a subject suffering fromatherosclerosis.

[0025] In yet another aspect, according to the invention there isprovided a method for preventing the mutagenic activity induced by freeradicals in a subject.

[0026] In still another aspect, the method of the present invention isuseful for treating a subject suffering from a tumor.

BRIEF DESCRIPTION OF THE FIGURES

[0027]FIG. 1 shows the dose-dependent protective effect of thecombination of the active ingredients on UVB-induced hemolysis (Example1).

[0028]FIG. 2 shows the dose-dependent protective effect of lipophiliccomponents of the combination of the active ingredients on UVB-inducedhemolysis (Example 2).

[0029]FIG. 3 shows the dose-dependent protective effect of hydrophiliccomponents of the combination of the active ingredients on UVB-inducedhemolysis (Example 2).

[0030]FIG. 4 shows the UVB-induced hemolysis: cooperative antioxidantinteraction between lipophilic and hydrophilic components of thecombination of the active ingredients (Example 3).

[0031]FIG. 5 shows the protective effect of the combination of theactive ingredients on RBC hemolysis induced by 120 min irradiation(Example 3).

[0032]FIG. 6 shows the effect of selenium yeast and sodium selenitesupplementation on keratinocytes viability (Example 4).

[0033]FIG. 7 shows the effect of selenium yeast and sodium selenitesupplementation on keratinocyte glutathione peroxidase activity (Example5).

[0034]FIG. 8 shows the effect of selenium yeast and sodium selenitesupplementation on glutathione peroxidase activity in keratinocytesexposed to UVB radiation (50 mJ/cm²) (Example 5).

[0035]FIG. 9 shows the effect of selenium yeast and sodium selenitesupplementation on cumene hydroperoxide (CuOOH)-induced cell death(Example 6).

[0036]FIG. 10 shows the effect of selenium yeast and sodium selenitesupplementation on CuOOH-induced intracelluar oxidative stress (DCFformation) (Example 7).

DETAILED DESCRIPTION OF THE INVENTION

[0037] In a preferred embodiment the method according to this inventionincludes the administration of vitamin E, beta carotene, an extract ofLycopersicum esculentum, vitamin C, an extract of Vitis vinifera and aselenium compound. Preferred selenium compounds are sodium selenite orselenium yeast.

[0038] The term “extract of Lycopersicum esculentum” as used hereinaboveor hereinbelow includes a dilute composition of lycopene. Said lycopenemay be obtained by extraction from plants, algae, fingi or geneticallymodified organisms or by synthesis. Preferably the extract from driedskin of tomatoes is directly utilized.

[0039] The term “extract of Vitis vinifera” as used hereinabove orhereinbelow includes a dilute composition procyandolic oligomers.Preferably the extract from the seeds of red vines is directly utilized.

[0040] In another preferred embodiment the method according to thisinvention includes the administration of:

[0041] (a) 5 to 20 parts, preferably 7.5 to 15 parts of vitamin E, inparticular vitamin E acetate or vitamin E succinate;

[0042] (b) 1 to 15 parts, preferably 2 to 10 parts of beta-carotene;

[0043] (c) 1 to 4 parts, preferably 1.5 to 3 parts of lycopene in theform of an extract of Lycopersicum esculentum obtained from dried skinof tomatoes.

[0044] (d) 2 to 60 parts, preferably 5 to 50 parts of vitamin C, inparticular the sodium salt thereof; and

[0045] (e) 1 to 30 parts, preferably 1.1 to 25 parts of Vitis viniferaextract in particular obtained from the seeds of red vines.

[0046] Preferably the lipophilic antioxidant and the hydrophilicantioxidant are administered in the presence of a pharmaceuticallyacceptable carrier. Suitable excipients are exemplified in: Remington:the science and practice of pharmacy. 19th ed. Easton: Mack Publ., 1995.

[0047] Most preferred carriers are selected from the group consisting ofnatural plant oils, fully or partially hydrogenated plant oils, inparticular soybean oil, fully or partially hydrogenated soybean oil,rape oil, peanut oil, lecithins, in particular soylecithin oregglecithin, plant phosphatides, in particular soyphosphatide andnatural waxes, in particular beeswax.

[0048] The compositions may be formulated as solids or solutions. Solidformulations maybe for preparation of a solution before injection.Preferably, the pharmaceutical compositions of the invention areadministered in the form of soft or hard gelatin capsule, tablet, coatedtablet, suppository or in the transdernal form. The dosage will beadjusted according to factors like body weight and health status of thepatient, nature of the underlying disease, therapeutic window of thecompound to be applied, solubility, and the like. It is within theknowledge of the expert to adjust dosage appropriately.

[0049] Accordingly, a further aspect of the present invention is the useof a composition of the invention in the preparation of a pharmaceuticalcomposition for the treatment of a subject suffering from pathologicalconditions being caused at least in parts to an overproduction of freeradicals like aging, inflammation, atherosclerosis or cancer.Furthermore, an aspect of the invention is a method of prevention and/ortreatment of inflammation, atherosclerosis or cancer, comprisingadministering an effective amount of a pharmaceutical composition of theinvention to a patient.

[0050] The objective of this study was to evaluate first in raterythrocytes the protective effect of the combination of the extracts ofVitis vinifera, Lycopersicon esculentum, vitamin C, vitamin E andbeta-carotene against the UVB-induced damage and the existence of acooperative interaction between its components, and then inkeratinocytes cell lines the ability of selenium to increase theendogenous enzymatic antioxidant defenses.

[0051] In detail the following investigations have been carried out:

[0052] (a) the dose-dependent ability of the combination of the extractof Vitis vinifera, the extract of Lycopersicon esculentum, vitamin C,vitamin E and beta-carotene, thereafter denominated in this text as“combination of the active ingredients,” to prevent hemolysis induced bylarge UVB doses (1.5-7.0 J/cm²);

[0053] (b) the dose-dependent ability of a mixture of the lipophilicantioxidants (Lycopersicon esculentum extract, beta-carotene+vitamin Eacetate) to inhibit hemolysis and lipid peroxidation in red cellmembranes;

[0054] (c) the dose-dependent ability of a mixture of the hydrophilicantioxidants (Vitis vinifera extract and vitamin C) to inhibit hemolysisand lipid peroxidation in red cell membranes;

[0055] (d) the protective effect on hemolysis and lipid peroxidation inred cell membranes by the association of the mentioned lipophilic andhydrophilic antioxidants;

[0056] (e) the ability of selenium yeast to release selenium in acellular system (keratinocytes): induction of glutathione peroxidase(GSH-PX) activity and cellular resistance to oxidative stress induced byphysical (UVB) or chemical (cumene hydroperoxide) cytotoxic agents.

EXAMPLES Materials and Methods

[0057] Chemicals

[0058] The organic solvents used in the experiments were of analyticalgrade (Carlo Erba, Milan, Italy). The active ingredients (Vitis viniferaextract, Lycopersicon esculentum extract, beta-carotene, lycopene, d,1-αtocopherol acetate, ascorbic acid, selenium yeast) were supplied byPharmaton SA, CH 6934 Bioggio, Switzerland. Cumene hydroperoxide(CuOOH), thiobarbituric acid (TBA), EDTA, sodium selenite (Na₂SeO₃),sodium azide (NaN₃), sodium pyruvate, 2′,7′-dichlorodihydrofluoresceindiacetate (DCFH-DA), DMEM (Dulbecco's modified Eagle's medium), phenolred free DMEM, L-glutamine, penicillinstreptomycin solution werepurchased from Sigma (Trimital, Milan, Italy); fetal calf serum andphosphate buffer solutions (PBS) from Hyclone (Belbio, Milan, Italy);H₂O₂ (30% v/v) from Fluka (Trimital, Milan, Italy); NADH, NADPH, GSH andGSH-reductase from Boehringer Mannheim Italy (Milan, Italy).

[0059] Apparatus

[0060] Spectrophotometric and fluorimetric studies were carried out in acomputer-aided Perkin Elmer Lambda 16 spectrophotometer and a computeraided Perkin Elmer LS50B luminescence spectrometer (Perkin Elmer, Monza,Italy).

[0061] UVB Irradiation of Erythrocytes (RBC)

[0062] Erythrocytes from male Wistar rats (Charles River, Calco, Italy),isolated and washed from white blood cells and platelets were suspendedin phosphate buffered saline, pH 7.4 (125 mM NaCI and 10 mM sodiumphosphate buffer (PBS)), stored at 4° C. and used until 4 days old. Forexperiments, an aliquot of the erythrocyte suspension was drawn from thestock, centrifuged at 1000×g for 5 min and 0.1 ml of the pellet dilutedto 50 ml with PBS obtain a 1% erythrocyte suspension (approximately15×10⁷ cells/ml).

[0063] 50 ml aliquots of the RBC suspension were placed in Pyrex glassdishes (14 cm diameter) transparent to exciting-light wavelengths>290 nmand exposed to UBV radiation for different time intervals. When theinhibitors were added to the RBC suspension, a pre-incubation period of30 min at 37° C. was applied. UVB radiation was carried out with aparallel bank of two Philips TL20W/12 fluorescence tubes (Sara s.r.l.,Castellanza, Italy) emitting a continuous spectrum between 280 and 320nm with a peak emission at 312 nm. Fluence rate at the site of cellirradiation (25 cm from UVB source) was of 0.85 mW/cm² and the UVB dosesemployed ranged from 1.5 to 7.0 J/cm² (exposure time 30-150 min), asmeasured with a Vilber Lourmat VLX-3W radiometer (UVB probe, 312 nm).

[0064] UVB-Induced Hemolysis

[0065] After irradiation, 2 ml aliquots of the RBC suspension werediluted 1:5 with buffered saline and hemolysis determinedturbidimetrically at 710 nm at 30 min intervals for 180 min. Percentageshemolysis were determined setting as a 100% hemolysis the absorbancevalue determined in RBC suspensions sonicated for 5 sec at 50% power.Mean values of 5 determinations were used for calculation.

[0066] Keratinocytes

[0067] The NCTC 2544 human keratinocyte cell line (Flow Laboratories,Irvine, UK) was cultured at 37° C. in DMEM supplemented with 10% fetalcalf serum 2 mM L-glutamine and antibiotics (100 U/ml penicillin and 0.1mg/ml streptomycin). Cultures were maintained at 37° C. in a humidified5% CO₂ atmosphere. For experiments, cells were seeded at a density of5×10⁴/cm² and cultured in 2 cm² cell plastic culture dishes until 80-90%confluence. Cell viability was determined by the trypan blue exclusionassay and by measuring the LDH leakage. Protein determinations wereperformed by a modified Lowry method, using bovine serum albumin as astandard.

[0068] Glutathione Peroxidase (GSH-PX) Activity

[0069] Cells grown were treated with sodium selenite (50, 500 nM) orselenium yeast (3.95 or 39.5 μg/ml corresponding to 50 and 500 nM Se)and incubated to 90% confluence (48 h). Before irradiation cellmonolayers were washed three times with prewarmed PBS and exposed to UVB(50 mJ/cm²), in the presence of PBS. After irradiation, PBS was replacedby serum-phenol red free DMEM (K-DMEM) and incubated for 24 h(sham-control cells were subjected to the same procedure without UVexposure). The media were discarded and the cells removed by gentlyscraping into 1 ml of 50 mM Tris-HCl buffer and lysed by sonication. Thecellular suspensions (0.1 ml aliquots) were analysed for GPX activity bymonitoring the decrease of absorbance at 340 nm for the rate ofdisappearance of NADPH in a thermostated (37° C.) spectrophotometer. GPXactivity was calculated using a molar extinction coefficient of NADPH of6.22×10³ cm⁻¹ m⁻¹ and expressed as mU/mg protein, where 1 U of enzymeactivity is defined as μmoles substrate consumed or product produced/minat 37° C. CUOOH-Induced Oxidative Stress

[0070] The intracellular oxidative stress induced by CuOOH and theprotective effect by selenium yeast was determined by measuring cellviability and peroxide content by using the oxidation-sensitivefluorescent probe DCF. Cells grown were treated with sodium selenite(50, 500 nM) or selenium yeast (3.95 or 39.5 μg/ml corresponding to 50and 500 nM Se) and incubated to 90% confluence (48 h). Stock solution ofDCFH-DA (3.34 mM) were prepared in ethanol, purged with nitrogen andstored at −20° C. in 500 μl aliquots. Cell monolayers were washed withprewarmed PBS and incubated with 10 μM DCFH-DA in PBS for 30 min at 37°C. Thereafter the medium was discarded and the cell monolayer washedthree times with prewarmed PBS to remove not-incorporated DCFH-DA andthen incubated with K-DMEM containing 0.5 mM CuOOH. Sham-control cellswere subjected to the same procedure without CuOOH exposure. After 24 hincubation, the media were discarded and the cells removed by gentlyscraping into 3 ml of PBS. The cellular suspension was then transferredinto 1 ml thermostated fluorescence cuvettes (25° C.) equipped with amagnetic stirrer and the increase of fluorescence intensity of the probe(λ_(exc) 502 nm; λ_(em) 520 nm; band widths 5 nm) recorded. Thefluorescence intensity values at all the observation times werecorrelated to protein content (means±S.D. of 6 determinations) andexpressed as % increase of fluorescence in respect to the controls(sham-control cells).

[0071] Statistical Analysis

[0072] Results are expressed as mean±S.D. of 5 independentdeterminations. Data were analysed by one-way analysis of variance(ANOVA), followed by the Tukey's test for statistical significance amonggroups and by Student's t-test (two tailed) for unpaired samples.Differences were considered significant when p<0.05. Statisticalanalysis was performed by using the Prism software package (GraphPadSoftware Inc., San Diego Calif., USA).

EXAMPLE 1 Protective Effect of the Combination of the Active Ingredients

[0073] Exposure of rat RBC to increasing UVB doses gives the typicalhemolysis curve reported in FIG. 1: the hemolytic process begins from 60min irradiation, corresponding to an UVB dose of approximately 3 J/cm²(8.5±3.2%) and reached the plateau between 90 (87.2±6.3%) and 120minutes (98.7±4.4%). The samples of the combination of activeingredients were prepared by mixing with PBS and vortexed; thehomogenous suspension obtained was further diluted in PBS to finalconcentrations ranging from 0.1-10 μg/ml. When unirradiated RBC wereincubated under aerobic conditions in the presence of the combination ofactive ingredients no significant hemolysis was observed within 3 h. Thecombination of active ingredients added to RBC suspension beforeirradiation, markedly and dose-dependently protected RBC fromUVB-induced hemolysis (FIG. 1), with a minimal effective concentrationof 0.1 μg/ml. At 5 μg/ml the hemolytic process was delayed by 30 min,and after 120 min irradiation the percentage hemolysis was only34.5±5.5% and at the highest concentration (10 μg/ml) RBC integrity wasmaintained until 150 min VB irradiation (7 J/cm²).The IC₅₀ value,calculated at 120 min, was 4 μg/ml.

EXAMPLE 2 Protective Effect of the Combination of Lipophilic andHydrophilic Antioxidants of the Combination

[0074] For a better understanding of the protective effect of thecombination of the active ingredients on UVB-induced hemolysis, we haveinvestigated the radical scavenging ability of a mixture of lipophilic(vitamin E acetate+beta-carotene+extract of Lycopersicon esculentum) andhydrophilic antioxidants (vitamin C+extract of Vitis vinifera).

[0075] Stock solutions of vitamin E acetate, beta-carotene and extractof Lycopersicon esculentum were prepared in THF under N₂ atmosphere andred light to prevent autooxidation and stored at −20° C. up to 1 week.The stability of each antioxidant was daily checked by UV spectroscopy.In order to maintain the same ratios between the antioxidants present inthe combination of active ingredients, a mixed stock solution of 7.04mg/ml for vitamin E acetate, 1.64 mg/ml for beta-carotene and of extractof Lycopersicon esculentum corresponding to 0.88 mg/ml lycopene wasprepared by dilution with THF. A serial dilution was made to give themixed lipophilic antioxidant working solutions at final concentrationsof 1.2, 2.4, 4.8, 9.6 μg/ml of RBC suspension (total lipophilicantioxidants). In all the experiments THF concentration was always 0.1%(v/v).

[0076] The same approach was used for hydrophilic antioxidants: a mixedstock solution of vitamin C (6.72 mg/ml) and extract of Vitis vinifera (2.8 mg/ml) was prepared in PBS and a serial dilution was made to givethe mixed hydrophilic antioxidant working solutions at 0.58, 1.16, 2.32,4.64, 9.28 μg/ml RBC suspension (total hydrophilic antioxidants).

[0077] As shown in FIGS. 2 and 3, both the lipophilic and thehydrophilic fractions of the combination of active ingredients are ableto prevent UVB-induced hemolysis, in a dose-dependent manner, althoughwith different potency. The greater activity of the hydrophilic mixtureis mainly due to the presence of the Vitis vinifera extract, which hasbeen shown in different experimental models in vitro to be a potentchain-breaking antioxidant (Maffei Facino R.; Carini M.; Aldini G.;Bombardelli E.; Morazzoni P.; Morelli R., “Free radicals scavengingaction and anti-enzyme activities of Procyanidines from Vitis vinifera—Amechanism for their capillary protective action”, Arzneim. Forsch./DrugRes. 44, 592-601, 1994) and to save endogenous vitamin E fromUVB-induced consumption, through recycling mechanism (hydrogentransferring reaction).

EXAMPLE 3 Cooperative Antioxidant Interaction Between Lipophilic andHydrophilic Antioxidants of the Combination

[0078] The cooperative antioxidant interaction between lipophilic andhydrophilic antioxidants of the combination was studied by usingconcentrations of each mixed working solution that give approximately20-30% inhibition at 120 min irradiation: 2.4 μg/ml for lipophilic(Vitamin E acetate 1.76 μg/ml, β-Carotene 0.41 μg/ml, lycopene 0.22μg/ml) and 0.58 μg/ml for hydrophilic (Vitamin C 0.4 μg/ml, extract ofvitis vinifera 0.18 μg/ml). The results are shown in FIGS. 4 and 5.Under these conditions, the rate of hemolysis was similar for both themixtures, the hydrophilic being slightly more active than the lipophilicone (20% vs 14% inhibition at 120 min). When the antioxidants werecombined, the rate of hemolysis was markedly slowered and the integrityof RBC was maintained up to 90 min. At 120 min the percentage inhibition(55±2.5%) was far beyond the sum of the % inhibitions (calculated:33.8%)observed with the individual antioxidant mixtures, to indicate asynergistic interaction. The protective effect elicited by a typicalcombination of hydrophilic/lipophilic antioxidants (Vitis viniferaextract+lycopene) did not differ significantly from that observed withthe combination of the hydrophilic ingredients (FIG. 5); by contrast,the association between vitamin E (1.76 μg/ml) and lycopene (0.22 μg/ml)gives a percentage inhibition of hemolysis significantly lower than thatobtained with the combination of the lipophilic ingredients (7.1% vs14%) (FIG. 5).

EXAMPLE 4 Effect of Selenium Yeast Supplementation on Cell Growth andCell Viability

[0079] In cultured keratinocytes, sodium selenite or selenium yeast hadno apparent growth-stimulating activity. Cell viability, determined LDHleakage (FIG. 6), was always over 95% and not significantly different inrespect to the sham-control cells at 50 and 500 nM Se doses.

EXAMPLE 5 Effect of Selenium Yeast Supplementation on GSH-PX Activity

[0080]FIG. 7 shows the effects of selenium yeast or sodium selenitesupplementation (equimolar Se doses) in control cells: with sodiumselenite maximal induction of GSH-PX activity was found at 50 nM (3.365fold increase vs controls; 91.44±10.37 vs 28.31±5.87 mU/mg protein), andno further increase was observed at 500 nM concentration. Selenium yeastwas less potent as GSH-PX inducer at the lower concentration (2.44-foldincrease; 63.05±5.51 mU/mg protein), but at the concentration 39.5 μg/ml(corresponding to 500 nM Se) the increase in the enzymatic activity wascomparable to that obtained with the equimolar dose of sodium selenite(90.11±10.35 mU/mg protein). These results clearly demonstrate thatselenium yeast dose-dependently releases selenium for enzyme activation.

[0081] GSH-PX activity was also determined in keratinocytes exposed toUVB irradiation (50 mJ/cm²), and the extent of enzyme induction byselenium yeast or sodium selenite was significantly enhanced incomparision to the control cells (FIG. 8), thus to indicate that alsounder oxidant imbalance selenium is still active.

EXAMPLE 6 Effect of Selenium Yeast Supplementation on CuOOH-Induced CellDeath

[0082] Cumene hydroperoxide is a strong promoter of free radicalreactions and incubation of keratinocytes with 500 μM CuOOH producedmore than 80% cell death 24 h after exposure (FIG. 9). In seleniumyeast-supplemented cells, cell death was markedly and dose-dependentlyrestrained, cell viability being 64±1.3% at the lowest dose (3.95 μg/ml)and not significantly different from controls cells (90±1.2%) at thehighest Se dose (39.5 μg/ml≈500 nM Se). The protection afforded by 50 nMsodium selenite was slightly greater, since cell viability was almostcompletely recovered.

EXAMPLE 7 Effect of Selenium Yeast Supplementation on IntracellularCuOOH-Induced Oxidative Stress

[0083] The intracellular oxidative status was determined by thesensitive fluorimetric DCF assay. The assay is based on the ability ofthe non-polar, non-fluorescent DCFH-DA to diffuse through the cellmembrane and to be deacetylated by cytosolic esterases to form thepolar, non-fluorescent dichlorodihydrofluorescein (DCFH). This last istrapped within the cytosol where, by reacting with reactive oxygenspecies generated by CuOOH, gives rise to the formation of thefluorescent derivative dichlorofluorescein (DCF).

[0084] Incubation of keratinocytes with 500 μM CuOOH results in asignificant accumulation of DCF (maximal after 24 h; more than 7-foldincrease), to confirm the spreading of an oxidative burst in the cytosolcompartment (FIG. 10). Pretreatment of the cells with selenium yeastcounteracts the oxidative insult, since a 60% reduction of DCF formationwas observed at the lower dose (3.95 μg/ml) and an almost completeprotection at 39.5 μg/ml (550 nM Se). Sodium selenite totally preventsDCF formation already at the lowest dose (50 Nm).

[0085] These results of these examples indicate that it is theassociation between lipophilic and hydrophilic antioxidants, which isthe object of the present invention that provides optimal cellprotection against the oxidative challenge of UVB. In other words, thereexists a synergistic interaction among antioxidants which explains thehigh degree of protection obtained with the combination of the activeingredients.

[0086] Since free radicals and reactive oxygen species (ROS) aregenerated in both the lipid and aqueous cell compartments, anantioxidant defense system as in the combination of the activeingredients, the object of the present invention, which can partition inboth lipid and aqueous environments, is highly suited for cellprotection.

[0087] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, this invention is not limited to the particularembodiments disclosed, but is intended to cover all changes andmodifications that are within the spirit and scope of the invention asdefined by the appended claims.

[0088] All publications and patents mentioned in this specification areindicative of the level of skill of those skilled in the art to whichthis invention pertains. All publications and patents are hereinincorporated by reference to the same extent as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference.

What is claimed is:
 1. A method of protecting a cell from damage causedby an overproduction of free radicals, said method comprisingadministering to said cell a therapeutically-effective amount of amixture comprising: (a) a lipophilic antioxidant comprising vitamin E,beta carotene, and an extract of Lycopersicum esculentum; and (b) ahydrophilic antioxidant comprising vitamin C and an extract of Vitisvinifera.
 2. The method of claim 1, wherein said mixture furthercomprises selenium.
 3. The method of claim 1, wherein said lipophilicantioxidant comprises: (a) 5 to 20 parts of vitamin E; (b) 1 to 15 partsof beta carotene; and (c) 1 to 4 parts of lycopene in the form of anextract of Lycopersicum esculentum.
 4. The method of claim 1, whereinsaid hydrophilic antioxidant comprises: (a) 2 to 60 parts of vitamin C;and (b) 1 to 30 parts of an extract of Vitis vinifera.
 5. The method ofclaim 1, wherein said vitamin E is vitamin E acetate or vitamin Esuccinate.
 6. The method of claim 1, wherein said vitamin C is thesodium salt of vitamin C.
 7. The method of claim 2, wherein saidselenium is sodium selenite or selenium yeast.
 8. The method of claim 1,wherein said mixture is administered to a cell in the presence of apharmaceutically acceptable carrier.
 9. The method of claim 8, whereinsaid pharmaceutically acceptable carrier is selected from the groupconsisting of natural plant oils, fully or partially hydrogenated plantoils, lecithins, plant phosphatides, and natural waxes.
 10. The methodof claim 9, wherein said pharmaceutically acceptable carrier is selectedfrom the group consisting of soybean oil, fully or partiallyhydrogenated soybean oil, rape oil, peanut oil, soylecithin,soyphosphatide, egglecithin, and beeswax.
 11. The method of claim 1,wherein said mixture is administered to a cell in the form of a soft orhard gelatin capsule, tablet, coated tablet, suppository, or in thetransdermal form.
 12. A method of protecting a patient from a disease,or treating a patient suffering from a disease, wherein said disease iscaused by an overproduction of free radicals, said method comprisingadministering to a patient a therapeutically-effective amount of amixture comprising: (a) a lipophilic antioxidant comprising vitamin E,beta carotene, and an extract of Lycopersicum esculentum; and (b) ahydrophilic antioxidant comprising vitamin C and an extract of Vitisvinifera.
 13. The method of claim 12, wherein said mixture isadministered to a patient in the presence of a pharmaceuticallyacceptable carrier.
 14. The method of claim 12, wherein said diseasecaused by an overproduction of free radicals is selected from the groupconsisting of aging, inflammation, atherosclerosis, and cancer.